According to mutations occurred in the passages during the Equine infectious anemia virus (EIAV) vaccine attenuation, a full length chimeric gene clone, pLGFD9-12, was constructed successfully at a backbone of clone pLGFD3-8 by substitution with LTR U3 region of a virulent EIAV strain. The pLGFD9-12 was used to transfect fetal donkey dermal (FDD) cells and passaged in donkey leukocyte (DL) culture. The cell cultures were monitored by RT-PCR, reverse transcriptase activity assay and real-time RT-PCR. The results of RT activity and RT-PCR were positive in the supernatant of cell cultures and viruses particles were also clearly observed under electron microscope. The replication of pLGFD9-12 chimeric viruses and its parental virus pLGFD3-8 has no obvious differences. The level of replication of the pLGFD9-12 chimeric clone cultured in DL cells was higher than that in FDD cells. The characteristics on virulence and replication ability of pLGFD9-12 will be evaluated in vivo.